ISOLATION AND MAINTENANCE OF ORAL L FORMSOF BACTERIA IN LIQUID MEDIA

ELEANOR GILMORE AND GEORGE W. BURNETT

Walter Reed Army Institute of Research, Washington, D. C.

Received for publication July 23, 1958

A variety of factors assist or cause the normalcells of a bacterial species to transform into andmaintain themselves as L organisms but asDienes and Sharp (1956) have stated: "it is astriking fact that transformation of bacteria toL forms has been observed to occur only onmedia solidified with agar." Even after L organ-isms have been maintained for some time onserum agar in the presence of a stabilizing agent,ordinarily they will revert to the normal cellulartype when transferred to any of the fluid mediaused for their cultural growth. In only a fewinstances have certain L organisms been grown inliquid media free of a transforming or stabilizingagent. L forms of Streptobacillus moniliformiswere grown in a liquid medium free of a trans-forming agent, but only after subculturing manytimes in the presence of the agent (Heilman,1941). Some L forms of Bacteroides and Salmo-nella species have been grown in broth containinga small amount of agar (Dienes and Weinberger,1951). Some, but not all, L strains of Proteusspecies were cultured as a thin film on the surfaceof serum broth containing penicillin (Tulasne,1950; Dienes, 1953) or in sterile urine and incasamino acid broth (Abrams, 1955). The Lstrains of Proteus species which have been grownin liquid media were isolated initially on thesurface of agar media containing penicillin.Penicillin was required to maintain such L strainsin subculture in liquid media, otherwise reversionoccurred. In some instances, initiation of growthof L organisms in liquid media required inocula-tion with agar blocks containing L colonies(Tulasne, 1950; Abrams, 1955). In other instancesit was necessary to add liquid media to the surfaceof a viable agar culture of the L organisms inorder to initiate growth (Dienes, 1953). Some ofthe stabilized L forms could be transferred seriallyin broth (Tulasne, 1950; Dienes, 1953; Abrams,1955), but others could not (Abrams, 1955).Failure of bacteria to change to or be maintainedas L forms in the absence of a transforming orstabilizing agent in liquid media does not seem to

be due to a lack of nutrients. Toxicity or properosmotic pressure have been suggested as impor-tant factors in the growth and maintenance of Lforms in liquid media. The study of L organismswould be greatly facilitated if they could begrown in liquid media. Accordingly, an attemptwas made to develop a liquid medium and aculture technique which would be optimal forisolation and maintenance of L organisms ofbacterial origin without the presence of a specifictransforming agent.

EXPERIMENTAL METHODS

Cultures. A pleuropneumonialike organism,PPLO strain H606, obtained from a virus mousebrain passage was used as the initial test organ-ism. After development of a suitable liquidmedium and technique for culture of the PPLOstrain, human saliva and residual material fromabout the crown of human teeth were used asthe source of PPLO and L organisms. In addi-tion, an unstable L form of Corynebacteriumspecies obtained from Wittler et al. (1956) and anL form of Gaffkya tetragena isolated from salivawere also used for determining the degree ofstability of L organisms in liquid media.

Culture media and techniques. All PPLO and Lstrains were grown routinely on PPLO agar(Difco). A comparison of PPLO or L growth wasmade on nineteen solid and fluid media, obtainedby varying the proportions of protein base, re-ducing substances, bacteriostatic compounds,and ascitic fluid or serum enrichment, underaerobic conditions and reduced oxygen tension.

Inoculation and transfer of the PPLO and Lorganisms from either agar to agar or agar tofluid culture was made by transferring an agarblock containing colonies of the organism duringmaintenance culture. For initial isolation of oralL microorganisms, 0.2 ml of human saliva wasinoculated into liquid media or spread on thesurface of solid media; variable amounts ofscrapings from human teeth were inoculated in asimilar manner. Agar cultures were incubated

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in candle jars at 37 C and broth cultures wereincubated at 37 C in normal atmosphere. Brothcultures were subcultured at regular intervalsonto PPLO agar (Difco) for identification andenumeration of colonies.

Staining methods. Ordinary bacterial cells werestained by Gram technique. Colonies of the Lforms on agar were stained by Dienes' vitalstaining technique (1939) and permanent impres-sion preparations were stained with Giemsastain according to the method of Klieneberger(1935), as modified by Salaman (1946). Unstainedpreparations and wet mounts were examined byphase microscopy.

RESULTS

Liquid media for isolation of L organisms. Inthe final development of a liquid culture medium,a combination of 50 per cent veal infusion brothand 50 per cent thioglycolate broth (v:v), con-taining 1 per cent serum fraction (Difco) or 25per cent ascitic fluid and 200 to 500 units ofpenicillin per ml was found to be most suitable

for the initial isolation of L forms from the oralcavity. The normal pH of 7.0 to 7.2 of veal-thioglycolate broth permitted good growth of theL organisms, in contrast to the pH of 7.8 and 8.0required for growth in most other media.Veal-thioglycolate broth contained approximately0.25 per cent agar. Serum fraction (Difco)or ascitic fluid was essential for growth of Lorganisms in veal-thioglycolate broth. Theaddition of from 200 to 500 units of penicillinper ml sufficiently inhibited most bacterial over-growth to permit transformation and propagationof the L organisms.

Standard procedure used for isolation of oral Lorganisms. The following procedures were de-veloped to obtain oral L organisms that could bepropagated and maintained in liquid or on solidmedia containing no known transforming agent.

Initial isolation. Two-tenths ml of saliva orvariable amounts of tooth scrapings were inocu-lated into veal-thioglycolate broth to which thepenicillin and ascitic fluid were added immedi-ately before inoculation. After 48 hr incubation

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\ .. . ...Figure 1. A diagramatic representation of the growth on a PPLO agar (Difco) plate inoculated from

an initial isolation culture of saliva in veal-thioglycolate broth. The center well (F) contained 0.15 mlof a solution containing 10,000 units of penicillin per ml. Normal bacterial colonies developed in zone A.Zone B contained bacterial colonies in which large bodies had formed. In zone C there were typical Lcolonies and zone D was composed of L colonies with many vacuoles and amorphous masses. Zone Eusually contained no bacterial or L colonies.

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at 37 C, a finely granular growth of L organismsusually developed in the midportion of theanaerobic zone of the veal-thioglycolate brothculture. Although such growth was relativelyabundant, it seldom remained viable for morethan 96 hr.At the same time that the veal-thioglycolate

broth was inoculated, 0.2 ml of the saliva ormaterial from about the teeth was spread on thesurface of each of two ascitic fluid-enrichedPPLO (Difco) agar plates, one without peni-cillin and the other containing approximately0.15 ml of a solution containing 10,000 units ofpenicillin per ml in a center well cut from theagar with a sterile cork borer. After 48 hr incu-bation in a candle jar at 37 C, PPLO and L col-onies could be detected on the surface of theplates with the aid of the lower power of themicroscope. The purpose of direct inoculationof the oral material onto agar was to indicatewhether PPLO were present in the inoculum andwhich of the bacterial species would possiblyform L organisms in veal-thioglycolate broth.Occasionally, sufficient numbers of bacteriatransformed into L forms on the penicillin-containing agar so that the various stages oftransformation could be observed in severalzones which depended upon the concentration ofpenicillin diffusing through the medium. Theconcentration of penicillin was often criticalupon initial plating which led to adopting thecenter well technique for incorporating gradientsof penicillin into the medium rather than addingpenicillin so that it is uniformly distributedthroughout the media (shown diagramaticallyin figure 1). Centripetally, as the concentrationincreased as in zone B, figure 1, bacterial coloniesdeveloped in which some of the bacterial cellswere transformed into large bodies (figure 2).There were typical L-type colonies but no ordinarybacterial colonies nearer to the center well in azone of increased penicillin concentration as inzone C, figure 1 (see also figures 3 and 4). Quitenear the center well, in the zone with a highpenicillin concentration (zone D, figure 1), Ltype colonies developed which contained manyvacuoles and amorphous masses (figure 5). Gen-erally no bacterial or L colonies could be foundimmediately around the center well, in the areaof very high penicillin concentration (zone E,figure 1). If L colonies developed on the peni-cillin-containing agar, subcultures to penicillin-free agar were made to recover and identify the

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Figure 2. An impression preparation (Giemsastain) taken from zone B of figure 1 showing bac-teria producing large bodies. In zone B, penicillinconcentration was too great for the developmentof normal bacterial cells and not enough for thedevelopment of typical L organisms (X909).

corresponding bacterial form. Repeated testingshowed that relatively few L colonies developedwhen material from the oral cavity was inoculateddirectly onto agar in comparison to the largenumber of L colonies obtained by initially cul-turing such material in veal-thioglycolate brothbefore subculturing on appropriate agar.

Subculture. After 48 to 72 hr incubation, 0.2ml of the original veal-thioglycolate broth cul-tures, pipetted from the area of growth in themidportion of the anaerobic zone, was trans-ferred to PPLO agar with 0.15 ml of a solutioncontaining 10,000 units of penicillin per ml in acenter well and to PPLO agar without penicillin.After such plates were incubated for 48 hr at37 C in a candle jar, numerous colonies developedon each plate which could be positively identifiedas L colonies by their morphological characteris-tics (Wittler et al., 1956), and by their stainingcharacteriestics. Inoculation of an agar blockcontaining only L colonies from the penicillin

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Figure 8. A view at the junction between zones B and C (figure 1 showing a bacterial colony, withsome L colonies growing around its edge. A typical L colony (a) can be seen growing at some distancefrom the bacterial colony (b) (X 440).

Figure 4. A typical L colony without a periph-ery from zone C and occasionally from zone D(figure 1), showing normal morphological struc-ture of L colonies derived from oral microorgan-isms (Giemsa stain, X912).

plate to media of similar composition but withthe penicillin concentration reduced from 10,000units per ml to 1000 units per ml usually pro-duced a stable L form after 2 or 3 subcultures.L organisms stabilized in this manner usuallydid not revert to ordinary bacterial cells whensubcultured in penicillin-free PPLO broth. How-ever, if the L strain was transferred from theoriginal penicillin-free agar plate and subcul-tured on penicillin-free agar, frequently there wasa gradual reversion of the L to the bacterialform.

Growth characteristics in subculture. At the endof 48 hr, 1 ml of the original veal-thioglycolatebroth culture was inoculated into each of twotubes, one containing veal-thioglycolate brothwithout penicillin and the other containing thesame broth with 200 to 500 units of penicillin perml. These broth subcultures were for the pur-pose of temporarily maintaining a viable L cul-ture should the organism fail to grow on othermedia, for there were some variations amongoral L strains as to their ability to propagate on

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Figure 5. An L colony from zone D (figure 1) containing many vacuoles and amorphous masses (Giemsastain, X 1500).

solid media. With some L strains, excellentgrowth was obtained during the first severalweeks of subculture in which inoculation was bythe agar block technique, after which time thecultures became more difficult to propagate.Growth could be maintained in some instancesby spreading 0.1 ml of 5 per cent hog gastricmucin on the surface of the agar before inocula-tion of the L strain. If mucin was applied, normalgrowth usually could be re-established after oneor two subcultures. Several strains were en-countered that produced L colonies when trans-ferred from veal-thioglycolate broth to agarmedia but which could not be subcultured onagar media, and for which mucin would notstimulate growth. These strains were obtainedfrom tooth scrapings from which a pure cultureof L organisms often grew upon initial isolation,the growth of ordinary bacterial cells being in-hibited by penicillin in the veal-thioglycolatebroth. From such strains, a stable form wasusually obtained after 2 or 3 passages in thisbroth containing penicillin.

If the L organisms from an initial veal-thio-glycolate broth culture were inoculated directly

into a liquid maintenance medium (PPLO broth),large bodies developed which neither reverted tothe bacterial form nor transformed to smallforms or granules. However, if an L strain, whichwas subcultured several times in the penicillinbroth, was transferred to the maintenance me-dium, small forms or granules in clumps propa-gated themselves. These small forms were similarto those which were observed when growth wasestablished in the maintenance medium by theinoculation of stabilized L colonies by the agarblock technique.

Establishment in maintenance media. Afterstabilization of the L strains by subculturingseveral times in media containing penicillin, theycould then be adapted to and maintained instable form on penicillin-free PPLO agar (Difco)and in penicillin-free PPLO broth (Difco) of thesame composition but with the agar removed bypassing through filter paper. Adaptation of thestabilized oral L organisms to PPLO broth wasaccomplished initially by transferring an agarblock containing a heavy concentration ofcolonies to 100 ml of broth that was incubated at37 C. Usually at some time between 7 to 14

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days incubation, growth could be observed as aslight turbidity upon shaking the culture, atwhich time 10 ml was transferred to 100 ml offresh broth. After subculturing several times atthe first indication of turbidity, the length of theincubation period was lessened until good growthwas obtained in 18 to 24 hr. Large numbers ofthe small elementary reproductive elements werepresent in the penicillin-free broth cultures,yielding only small L colonies when subculturedon appropriate agar.When the L strains were once established in

PPLO broth, cultures were viable after weeks ofstorage at refrigerator temperatures. An exam-ination of the L growth in such PPLO mediacultures revealed a predominance of small bodiesin colony-like clumps. When the small bodiesoccurred singly they exhibited considerableBrownian movement.

Stabilization of L organisms with veal-thio-glycolate broth. When isolated in the manner de-scribed, the oral L organisms seldom reverted tothe ordinary bacterial form in broth after two orthree passages in the presence of the transformingagent. The degree of stability of these L cul-tures can be illustrated by our experience withthe L forms of G. tetragena isolated from saliva

with broth (figure 6). In the stable form, the Lstrain was maintained for two years on bothPPLO agar (Difco) and in PPLO broth (Difco)cultures. During this time, various media combi-nations and variable physical conditions whichmight be expected to cause the L strain to revertwere tested but the organism remained in a stablecondition until transferred to PPLO broth (BBL)which had not been used previously to culturethis L strain. The PPLO broth (BBL) differedfrom the previously used PPLO medium (Difco)in being compounded with a different type ofpeptone. When initially placed in PPLO broth(BBL), growth was slower than with the Difcomedium. Upon continued subculture in PPLObroth (BBL), the strain lost its stability andgradually reverted to the normal cellular form.At the beginning of the transformation, numerouslarge forms appeared, gradually passing to atransitory stage in which the elements weremostly filaments, and finally grew as swollenforms mixed with normal bacterial cells (figures7 to 10). The normal bacterial cells recoveredfrom these cultures was a strain of G. tetragena,identical with the gram-positive coccus isolatedtwo years previously. When maintained on PPLObroth (Difco), the L strain of G. tetragena did

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Figure 7A. A clump of small elements from a stable L form of Gaffkya tetragena at the time when itbegan to lose its stability. In the clump at (a), can be seen the typical small elements of the stable Lform. The scattered dark masses seen at (b) and elsewhere represent the beginning transformation of theL strain to its bacterial form. Growth was in PPLO broth (BBL). (Light phase microscopy, X 490.)

Figure 7B. The same culture as in figure 7A but viewed with dark phase microscopy. Ring forms aremore evident than when viewed with light phase microscopy (X 490).

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Figure 8. The results of further incubation ofthe reverting L form inPPLO broth (BBL) showingdevelopment of filament-like structures. Ringforms and small elements are still quite evidentat this stage (X970).

not revert to the gram-positive coccal form butremained indefinitely in the L form.The production of stable L strain by culturing

in broth is further demonstrated with an L strainof Corynebacterium species which was isolatedalong with a staphylococci and a typical PPLOstrain in another laboratory from a case of non-

specific urethritis. In the previous study of

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Figure 9 A transitory stage of oral L organismsof approximately 24 hr in PPLO broth (BBL) inwhich is seen filament-like structures with irregu-lar larger bodies enmeshed in the filaments (X915).

Wittler et al. (1956), the typical PPLO revertedto the ordinary cellular form of the Coryne-bacteriutm species in HeLa cells when a filtrateof the staphylococcus was added to the culture.The corynebacteria originally did not transformto the L type. After several years of subculturing,they grew as a mixture of bacterial and L formson agar containing penicillin and as the bacterialform only on agar without penicillin. At the timeof testing, this organism grew as half bacterial

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and half L type colonies. The mixture of bac-terial and L forms was scraped from the surfaceof the medium with a wire loop and inoculatedinto veal-thioglycolate broth. After 48 hr incu-bation, the culture was inoculated onto PPLOagar with and without penicillin. Numerouscolonies with the typical morphology of PPLOrather than L type colonies developed on bothplates (figure 11). Continued subculture on peni-cillin-free PPLO broth and agar did not resultin a reversion to the bacterial form, indicatingthat the strain had become stabilized duringthe culture procedure.

DISCUSSION

Figure 10. A culture of Gaffkya tetragena aftercomplete reversion from L to bacterial formsshowing normal size packets of cocci and swollencocci (Gram stain, X912).

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Figure 11. The typical pleuropneumonialikecolonies of a Corynebacterium species after trans-forming in veal-thioglycolate broth and subcul-turing on PPLO agar (Difco) (X512).

A medium and set of conditions have beenfound by which certain of the L organisms fromthe oral cavity and elsewhere can be transformed,isolated, and stabilized for growth in liquidmedia without the presence of a known trans-forming agent. By the use of this technique,which consists essentially of initial passage ofthe saliva sample or tooth scrapings through a

composite broth before subculturing on peni-cillin-containing agar, the number of oral Lorganisms isolated was greatly increased over

that obtained with other methods. Once ob-tained, such organisms were easily stabilized inthe L form. The L forms most frequently iso-lated from the oral cavity by this procedure were

derived from gram-positive cocci; only a fewgram-positive rods were obtained. While thetechnique of stabilization has been successfullyapplied to other bacterial species, this methodmay not be adequate for isolation and perhapsstabilization of the L forms of certain bacterialspecies.

Veal-thioglycolate broth has several peculiarcharacteristics with regard to growth of oral Lforms. Although the broth supported an abun-dant growth of L organisms, veal-thioglycolateagar would not support the growth of any L or-

ganisms. Oral L forms could be isolated andgrown easily in veal-thioglycolate broth, butcultures were usually not viable for more than96 hr. It is not likely that the broth sustains therapid growth of the L forms simply because ofits nutritional qualities. Furthermore, the hydro-gen ion concentration of the medium is not op-

timal according to previous concepts. On theother hand, hydrogen ion concentration could bea reasonable explanation for the short period

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during which the L forms are viable in the broth.When a broth culture only 48 hr old is inoculatedinto PPLO broth, growth occurs quickly asevidenced by the rapid appearance of turbidity,whereas when PPLO broth is inoculated by agarblock technique several weeks are required toinitiate growth which is shortened, however,with each subculture.

In addition to composition and physical prop-erties of media, it might be suspected from theresults obtained in the present investigationthat the stability and perhaps the ability ofpropagation of L forms is also related in someway to their rate of growth or reproduction.Furthermore, when the agar block techniquewas used to inoculate L forms in broth, the sta-bility of the L form was increased as the lengthof the incubation period decreased. In the in-stance described in which the L form of G.tetragena reverted to the bacterial form after twoyears of stability, transformation from the L tothe bacterial form was associated with a slowingof the growth rate. It has been our observationthat stability of L forms in broth depends, inpart, upon maintenance of the growth rate andearly subculture of the strain, especially in theabsence of penicillin.

SUMMARY

A liquid medium and culture technique wasdevised for the primary isolation and subcultureof oral L organisms, derived principally fromcocci, without the presence of a transformingagent. By initially culturing material from theoral cavity in a composite veal-thioglycolatebroth for no longer than 96 hr, numerous L or-ganisms could be propagated on suitable agarand in liquid media. Upon subculturing two orthree times on penicillin-containing media, the

L forms became sufficiently stabilized to growindefinitely in liquid media containing no trans-forming agent.

REFERENCES

ABRAMS, R. Y. 1955 A method of culturing Lforms in liquid media. J. Bacteriol., 70,251.

DIENES, L. 1939 "L" organisms of Klienebergerand Streptobacillus moniliformis. J. Infec-tious Diseases, 65, 24-42.

DIENES, L. 1953 Some new observations on theL forms of bacteria. J. Bacteriol., 66, 274-279.

DIENES, L. AND SHARP, J. T. 1956 The role ofhigh electrolyte concentration in the produc-tion and growth of L forms of bacteria. J.Bacteriol., 71, 208-213.

DIENES, L. AND WEINBERGER, H. J. 1951 TheL forms of bacteria. Bacteriol. Revs., 15,245-288.

HEILMAN, F. R. 1941 A study of Asterococcusmuris (Streptobacillus moniliformis). I.Morphological aspects and nomenclature. J.Infectious Diseases, 69, 32-44.

KLIENEBERGER, E. 1935 The natural occurrenceof pleuropneumonia-like organisms in appar-ent symbiosis with Streptobacillus monili-formis and other bacteria. J. Pathol. Bacte-riol., 40, 93-105.

SALAMAN, M. H. 1946 Isolation of organisms ofpleuropneumonia group from genital tract ofmen and women. J. Pathol. Bacteriol., 58,31-35.

TULASNE, R. 1950 Quelques donn6es nouvellessur la formation et les caracteres de culturedes formes L du Proteus vulgaris. Compt.rend. soc. biol., 144, 1200-1203.

WITTLER, R. G., CARY, S. G., AND LINDBERG, R.B. 1956 Reversion of a pleuropneumonia-like organism to a Corynebacterium duringtissue culture passage. J. Gen. Microbiol.,14, 763-774.

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